Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures

Mutation Research 498 (2001) 89–98

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures Hilada Nefic∗ Department of Genetics, Faculty of Science Sarajevo, Zmaja od Bosne 33-35, Sarajevo 71000, Bosnia and Herzegovina Received 20 March 2001; received in revised form 3 July 2001; accepted 5 July 2001

Abstract Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 ␮g/ml) had a limited antimutagen effect on cisplatin (0.5 ␮g/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Vitamin C; Cisplatin; Chromosomal aberrations; Anticlastogen

1. Introduction Vitamin C is an antioxidant and strong free radical scavenger [1–3]. Its antioxidant activity is useful for protection of cellular macromolecules, particularly DNA, from oxidative damage induced by different agents. Vitamin C also possesses DNA damaging and mutagenic activity inducing DNA strand breakage [4], increasing the frequency of sister-chromatid exchanges (SCEs) in cells [5,6], increasing the number of somatic mutations [7,8]. However, Vitamin C is not an active mutagen itself. Its mutagenicity is mediated by oxy radicals, appearing during oxidation of Vitamin C by molecular oxygen [1,7,9]. ∗ Tel.: +381-33-646-175; fax: +381-33-649-359. E-mail address: [email protected] (H. Nefic).

Cisplatin is an effective antitumour drug but it has serious side effects on nontumour cells, including free radical generation [10]. Cisplatin produces intra- and interstrand DNA cross-linking effects [11–14]. This cytostatic induces chromosomal aberrations in cultured mammalian cells [15–17], mouse bone marrow cells [16] and peripheral blood lymphocytes in patients [18] and sister-chromatid exchanges in human lymphocytes in vitro [16,19] and in vivo [16]. The frequency of occurrence of cells with micronuclei is increased by cisplatin in human lymphocytes in vitro [17,20] and in erythrocytes in bone marrow cells of mice [21]. Many in vivo assays found that Vitamin C successfully reduced the clastogenic effects of many antitumour agents [22–24]. Vitamin C inhibited tumour cell growth, binding of the active carcinogenic

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metabolite to cellular DNA [25]. Besides the antimutagenic and anticlastogenic properties of Vitamin C, it is also known that this vitamin has mutagenic and clastogenic effects. As an antioxidant, Vitamin C may also have potential anticancer activity, but its possible role and its mechanism of action in cancer prevention has not been clearly established. On the basis of data of experimental studies presented in literature Cozzi et al. [26] concluded that Vitamin C has anticarcinogenic and chemopreventive activities although the results of these studies are often conflicting. Osmak et al. [27] also found that Vitamin C may inhibit the growth of some tumour cells in vitro and in vivo. DNA is exposed to oxidative damage from free radicals generated during the normal metabolism of a cell and also during the metabolic activation of some mutagenic chemicals. In living organisms, the antioxidant system inactivates oxy radicals before their interaction with DNA. Antioxidants such as glutathione, urea, Vitamins E and C, or enzymes such as superoxide dismutase, catalase and glutathione peroxidase display antimutagenic and anticarcinogenic properties by protecting the cells from oxidative damage. If natural dietary antioxidants protect against endogenous oxidative DNA damage, it can be assumed that they should protect against many agents that can cause genetic damage through free radical mechanisms. This is important because certain genetic damage might be prevented by such antioxidants. Identification and analysis of agents with anticlastogenic activity that reduce the frequency of chromosomal aberrations and possibility of practical application of natural protectors against the clastogenic (and mutagenic/carcinogenic) action of chemical mutagens have great importance. In this study, the protective effects of Vitamin C on cisplatin induced chromosomal aberrations have been determined in human peripheral lymphocyte cultures by cytogenetic analysis (cytogenetics test of chromosome aberrations in vitro).

Vitamin C (PLIVIT® C; Pliva, Zagreb) was used as a free radical scavenger to investigate the effects of this vitamin on the number and type of cisplatin induced chromosome aberrations. 2.2. Cytogenetics test of chromosome aberrations in vitro The test of chromosome aberrations was modified according to Moorhead et al. [28]. Blood samples were obtained from six healthy volunteers, three females and three males aged 22–61 years. The heparinised human peripheral blood was cultured in medium RPMI-1640 (Sigma Chemical Co., St. Louis, MO) and supplemented with 20% fetal calf serum (Sigma Chemical Co.). As a consequence of the mitogenic effect of phytohemagglutinin (10 ␮g/ml) (PHA, Sigma Chemical Co.), lymphocytes were stimulated to division. The cultures were incubated at 37◦ C and harvested at 72 h after PHA stimulation. Cisplatin 0.5 ␮g/ml culture medium and Vitamin C 10 and 100 ␮g/ml culture medium were used. They were dissolved in water for injections. These concentrations of preparations were chosen on the basis of literature data and preliminary tests [17,29]. For each concentration of Vitamin C three cultures were established. For simultaneous treatment, cisplatin and Vitamin C were added to the cultures together 48 h after PHA stimulation and left until harvest. Vitamin C was added to the cultures 1 h prior to cisplatin as pre-treatment and 1 h after cisplatin as post-treatment. Treatment with cisplatin alone and Vitamin C alone, 48 h after PHA stimulation were also performed previously. Cells were arrested in metaphase by the addition of colchicine at a final concentration of 0.4 ␮g/ml culture medium and incubated for 2 h. Chromosome preparations were stained with 10% Giemsa for 10 min. A control untreated culture of lymphocytes was established as well. 2.3. Scoring of slides

2. Materials and methods 2.1. Chemicals Cisplatin (CISPLATIN Lösung Medac; Pharmachemie B.V. Haarlem/Holland) was used as a typical clastogen for inducing chromosome aberrations and

The mitotic index was determined by counting the number of mitotic cells in 1000 cells per culture for a total of 6000 cells per treatment and control. In order to obtain the frequencies of chromosomal aberrations, 100 metaphases from each culture were examined for a total of 600 cells per each treatment and control.

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Numerical and structural chromosome aberrations were analysed separately. Gaps were noted but not included in the statistical analysis. 2.4. Statistical analysis The value of the mitotic index, the number of aneuploid mitoses, the number of metaphases with structural chromosome aberrations, the total number of structural aberrations and their types and the frequency and number of aberrations per cell induced by cisplatin in comparison with the control culture and the culture treated with cisplatin alone were analysed statistically by the chi-square test (χ 2 -test).

3. Results The results obtained for this study are shown in Tables 1–3. Table 1 shows the effect of Vitamin C (the concentrations of 10 and 100 ␮g/ml culture medium) on the mitotic index, the number of chromosomal aberrations (numerical and structural) and the frequency of abnormal metaphases in human lymphocyte cultures treated with cisplatin (the concentration of 0.5 ␮g/ml culture medium). Treatment with Vitamin C alone at the concentration of 10 and 100 ␮g/ml culture medium did not change the mitotic index value notably when compared with the untreated control. The cultures treated with cisplatin alone had a statistically significant reduction in mitotic index (P < 0.001), also in the pre-treatments and post-treatments with Vitamin C and differences were statistically significant, P < 0.001. In the simultaneous treatments with cisplatin and Vitamin C the mitotic index of human lymphocytes changed significantly in comparison with control cultures (Vitamin C 10 ␮g/ml, P < 0.05 and 100 ␮g/ml, P < 0.001), but when compared with cultures treated with 0.5 ␮g/ml cisplatin alone, the mitotic index of these cultures were much higher (P < 0.001). Vitamin C alone, at the concentrations of 10 and 100 ␮g/ml, did not induce numerical chromosome aberrations significantly and cisplatin 0.5 ␮g/ml in the absence and the presence of Vitamin C, when compared with controls, caused a significant increase in aneuploid mitoses frequency (for all the treatments P < 0.001). Vitamin C did not show a clastogenic

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effect at the concentrations rested. Cisplatin at a concentration of 0.5 ␮g/ml produced a highly statistically significant increase in the number of abnormal metaphases and the total number of structural chromosome aberrations when compared with the untreated control (P < 0.001). Statistical analysis showed that there were also significant differences in the frequency of chromosomal and chromatid aberrations and abnormal metaphases between the control and cells treated with cisplatin and Vitamin C in the pre-, simultaneous and post-treatments (for all the treatments P < 0.001). However, Vitamin C showed an anticlastogenic effect against cisplatin induced damage at these treatments. In the presence of Vitamin C, the number of chromosomal aberrations (numerical and structural) statistically significantly decreased when compared with the culture treated with cisplatin at a concentration of 0.5 ␮g/ml alone (P < 0.001). The number of abnormal metaphases was markedly decreased in all the treatments in comparison with the culture treated with cisplatin alone, P < 0.001 (except in the pre-treatments: Vitamin C 10 ␮g/ml, P < 0.05 and 100 ␮g/ml, P < 0.005). The influence of Vitamin C on cisplatin induced chromosome aberrations was most effective in the simultaneous treatments. The types of structural chromosome aberrations and their frequency in human lymphocytes induced by cisplatin (at the concentration of 0.5 ␮g/ml) in the absence and the presence of Vitamin C (at the concentrations of 10 and 100 ␮g/ml) are presented in Table 2. The structural chromosomal aberrations induced by cisplatin in cultures of human lymphocytes in the absence and the presence of Vitamin C were chromosome and chromatid breaks, acentric fragments and minutes and chromatid rearrangements such as triradials, quadraradials or more complex exchanges. The number of chromosomal rearrangements and chromosomal and chromatid breaks in cultures treated with cisplatin in the presence of Vitamin C decreased, while the number of acentric fragments and minutes increased when compared with the culture treated with cisplatin alone. Table 3 gives results of analysis for the number of aberrations per cell in human lymphocyte cultures treated with cisplatin and/or Vitamin C.

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In the presence of Vitamin C, cisplatin induced increase in the number of cells with one and two aberrations and decrease in the number of cells with more aberrations compared to the cisplatin treated cultures.

4. Discussion Concentrations of Vitamin C used in the present experiments were 10 and 100 ␮g/ml and at these concentrations Vitamin C did not change the mitotic index value notably and did not have clastogenic effects. Cisplatin showed strong reduction in the mitotic index of human lymphocytes at the test concentration of 0.5 ␮g/ml and markedly induced chromosome aberrations in cultured human lymphocytes. However, the inhibitory effect of cisplatin on the mitotic activity of human lymphocytes was statistically significant decreased with simultaneous treatment by Vitamin C. Chromosome aberrations (numerical and structural) induced by cisplatin in human lymphocyte cultures decreased markedly in the pre-treatment, simultaneous and post-treatment with Vitamin C. However, the number of these aberrations was not reduced to the control number of chromosome aberrations. Vitamin C was most efficient in the simultaneous treatment. The results of both treatments with Vitamin C indicated that the number of aberrant metaphases were statistically significantly decreased in the pre-treatment, simultaneous and post-treatment of cisplatin with Vitamin C. Also, Vitamin C was most effective in the simultaneous treatments. The results obtained for this study suggest that Vitamin C decreases the number of chromosome aberrations induced with cisplatin, but it can not completely protect cells from damage. At the test concentrations used, Vitamin C has a limited antimutagen effect to cisplatin induced damage of genetic material. The antimutagen effect involves an anticlastogenic effect of Vitamin C and its capability of decreasing the number of aneuploid mitoses. Also, Vitamin C reduces the inhibitory effect of cisplatin on the mitotic activity of lymphocytes in the simultaneous treatment. Vitamin C shows most efficient anticlastogenic effect in the simultaneous treatments with cisplatin.

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Vitamin C also has in vivo anticlastogenic effects against chromosomal damage and number of abnormal metaphases induced by cisplatin in rodents [24,30–32]. Vitamin C successfully reduces the clastogenic effect of many other antitumour drugs like cyclophosphamide [33–36], doxorubicin [23,29] and bleomycin [34,37,38]. Under different conditions, Vitamin C acts as either a radical scavenger or a pro-oxidant producing hydrogen peroxide and free radicals by autoxidation [1,39]. In human lymphocytes, treatment with Vitamin C has a modifying effect on bleomycin mutagenicity [39]. Also, Vitamin C reduces the chromosome aberrations induced by trenimon [33,34], H2 O2 [40], ethyl methanesulfonate [41–43], levodopa, noradrenalin and dopamine [44] and pesticides [45] and SCEs induced by cyclophosphamide, mitomycin C [46], thiotepa, l-ethionine [47], oxygen radicals [39,48] and H2 O2 [26]. Vitamin C decreases the frequency of micronucleus formation induced by cisplatin [30], bleomycin [38] and doxorubicin [49] and inhibits the gene mutations induced by aflatoxin B1 [50], malonaldehyde and ␤-propiolactone [51], ethyl methane sulfonate [43] and tetracycline hydrochloride [52]. On the other hand, Vitamin C improves the antineoplastic activity of doxorubicin, cisplatin and paclitaxel in human breast carcinoma cells in vitro [53]. Park [54] shows that in vitro human leukemic-colony forming cells (L-CFC) growth suppression by Vitamin C is observed in one-sixth of leukemic patients and L-CFC enhancement in one-third of patients. There is evidence that Vitamin C enhances the gene mutations induced by N-methyl-N -nitro-N-nitrosoguanidine [5] and SCEs induced by thiotepa and l-ethionine [47] and benzo[a]pyrene [55]. Odin [56] shows that continual peroral consumption of large amounts of Vitamin C over a long time period is able to significantly decrease the number of chromosomal breaks induced in human lymphocytes by various xenobiotics. Similar results obtained Duthie et al. [57] show that supplementation of the diet with Vitamin C results in a highly significant decrease in endogenous oxidative base damage in the DNA of lymphocytes of patients, and lymphocytes of antioxidant-supplemented subjects show an increased resistance to oxidative damage in vitro. However, Vitamin C does not affect the frequency of aberrations in lymphocytes workers occupationally exposed to cytostatic drugs [58].

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The exact mechanism of action of Vitamin C is still unclear. The mechanism of its antimutagenicity is mainly connected with its free radical scavenging properties and to a lesser extent with its nucleophilicity. The protective role of Vitamin C observed here suggests that Vitamin C has some antioxidative effect on damage induced by free radicals generated during the metabolic activity of cisplatin in cultured human somatic cells. According to Gebhart [59] anticlastogenesis is an essential part of antimutagenesis and also of anticarcinogenesis. These data indicate that certain mutations and human cancers might be prevented by anticlastogenic (antimutagenic/anticarcinogenic) agents.

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